Stenotrophomonas goyi sp. nov., a novel bacterium associated with the alga Chlamydomonas reinhardtii

Background A culture of the green algae Chlamydomonas reinhardtii was accidentally contaminated with three different bacteria in our laboratory facilities. This contaminated alga culture showed increased algal biohydrogen production. These three bacteria were independently isolated. Methods The chromosomic DNA of one of the isolated bacteria was extracted and sequenced using PacBio technology. Tentative genome annotation (RAST server) and phylogenetic trees analysis (TYGS server) were conducted. Diverse growth tests were assayed for the bacterium and for the alga-bacterium consortium. Results Phylogenetic analysis indicates that the bacterium is a novel member of the Stenotrophomonas genus that has been termed in this work as S. goyi sp. nov. A fully sequenced genome (4,487,389 base pairs) and its tentative annotation (4,147 genes) are provided. The genome information suggests that S. goyi sp. nov. is unable to use sulfate and nitrate as sulfur and nitrogen sources, respectively. Growth tests have confirmed the dependence on the sulfur-containing amino acids methionine and cysteine. S. goyi sp. nov. and Chlamydomonas reinhardtii can establish a mutualistic relationship when cocultured together. Conclusions S. goyi sp. nov. could be of interest for the design of biotechnological approaches based on the use of artificial microalgae-bacteria multispecies consortia that take advantage of the complementary metabolic capacities of their different microorganisms.


Introduction
The first described species of the Stenotrophomonas genus was S. maltophilia, which was a Gram-negative bacterium originally named as Pseudomonas maltophilia, and later transferred in 1993 to the new genus Stenotrophomonas, which was solely composed of S. maltophilia.In 2001, this species was moved to the genus Xanthomonas before it was finally moved back again in 2017 to its own genus when Stenotrophomonas pictorum was identified (Ryan et al., 2009;Wei et al., 2021).Currently, Stenotrophomonas is a genus comprising at least 19 validated species (https://lpsn.dsmz.de/genus/stenotrophomonas) (Parte et al., 2020).However, the molecular taxonomy of the genus is still somewhat unclear, and all its members are considered as "orphan species".All Stenotrophomonas spp.have shown intraspecific heterogeneity with high phenotypic, metabolic, and ecological diversity (Ryan et al., 2009).
The main reservoirs of Stenotrophomonas spp.are soil and plants, although they are ubiquitously present in different environments, including opportunistic human pathogens such as S. maltophilia (Ryan et al., 2009).
Stenotrophomonas spp.show promising potential for different biotechnological applications.Some Stenotrophomonas spp.are of interest to agriculture due to their ability to promote growth in different plant species.Some Stenotrophomonas spp.are even capable of establishing symbiotic relationships with plants.This plant growth promotion is related to the capacity of some Stenotrophomonas spp. to produce the plant growth hormone indole-3-acetic acid (IAA), fix nitrogen, oxidate elemental sulfur (S) to sulfate, or biocontrol plant pathogens (Banerjee and Yesmin, 2008;Park et al., 2005;Ryan et al., 2009;Suckstorff and Berg, 2003).
Moreover, they are also considered good candidates for bioremediation due to their tolerance to heavy metals and capability to metabolize a large variety of organic molecules, including phenolic and aromatic compounds (Liu et al., 2007;Mora-Salguero et al., 2019;Pages et al., 2008;Ryan et al., 2009).Finally, some Stenotrophomonas spp.can synthetize useful bioproducts such as antimicrobial and enzymes of biotechnological interest (Rivas-Garcia et al., 2022;Wolf et al., 2002).
Here we report the genome of Stenotrophomonas goyi sp.nov.isolated from a contaminated microalgae (Chlamydomonas reinhardtii) culture.This alga culture was simultaneously contaminated with S. goyi, Microbacterium forte (Fakhimi et al., 2023a) and Bacillus cereus.The metabolic interactions established between these four microorganisms are analyzed and discussed in a related publication where the ability of this multispecies consortium to sustain hydrogen production is highlighted (Fakhimi et al., 2023a).

Methods
Isolation of Stenotrophomonas goyi sp.nov.This study took place at Campus Universitario de Rabanales, Cordoba, Spain.S. goyi sp.nov.where it was isolated from a fortuitously contaminated Chlamydomonas reinhardtii culture in the laboratory.Initially, the Chlamydomonas reinhardtii culture was simultaneously contaminated with three different bacteria (Fakhimi et al., 2023a).Individual members of this bacterial community were isolated by sequential rounds of plate streaking in Yeast Extract Mannitol (YEM) medium (handmade in our lab, described here), until three different types of bacterial colonies were visually identified.Colonies were grown separately, and the subsequent isolated DNA was used for PCR-amplification of their partial RNA 16S sequences.After sequencing, the three independently isolated bacteria were identified as members of the genus Microbacterium, Stenotrophomonas, and Bacillus (Fakhimi et al., 2023a).

REVISED Amendments from Version 2
A second accession number has been provided for the repository of Stenotrophomonas goyi sp.nov.The bacteria is now deposited in two internationally recognized culture collections.
The use of italics within the name of Stenotrophomonas goyi sp.nov.has been homogenized.

Two references have been updated.
Any further responses from the reviewers can be found at the end of the article completeness was checked by BUSCO 3.0.2(RRID:SCR_015008) (Manni et al., 2021) and was 94.6% complete, with 94.6% of the genome single copy and 0.0% duplicated.Any other prokaryotic contamination was discarded using ContEst16S 1.0 (RRID:SCR_000595) (Lee et al., 2017).

Coculturing algae and bacteria
Chlamydomonas cells were cultured for 3-4 days in TAP medium until mid-logarithmic growth phase, harvested by centrifugation (5.000 rpm for 5 min) and washed twice with fresh MM.Bacterial batch-cultures were incubated in TYM or LB medium until the Optical Density at 600 nm (OD 600 ) reached 0.8-1, then harvested by centrifugation (12.000 rpm for 5 min) and washed twice with fresh MM.Algae and bacteria were cocultured in 250 mL flasks containing 100 mL of the corresponding medium.Alga-bacterium cocultures were set to initial chlorophyll concentration of 10 μgÁmL À1 for the alga and an initial OD 600 of 0.1 for the bacterium.Algal and bacterial monocultures were used as controls.All cultures were incubated at 24°C with continuous agitation (120-140 rpm) and under continuous illumination (80 PPFD).

Determinations of algal and bacterial growth
The algal growth was assessed in terms of chlorophyll content.Chlorophyll measurements were done by mixing 200 μL of the cultures with 800 μL of ethanol 100%.The mix was incubated at room temperature for 2-3 min, and afterward centrifuged for 1 min at 12.000 rpm.The supernatant was used to measure chlorophyll (a + b) spectrophotometrically (DU 800, Beckman Coulter) at 665 and 649 nm (Wintermans and de Mots, 1965).
Bacterial growth in monocultures was estimated spectrophotometrically in terms of OD 600 evolution (DU 800, Beckman Coulter).However, estimation of the bacterial growth in cocultures required bacterium cells separation from the alga cells.To do this, a customized Selective Centrifugal Sedimentation (SCS) approach was used.This approach consisted in finding the centrifugation parameters that led to maximize algal cell sedimentation while minimizing bacterial cell sedimentation (Torres et al., 2022).Thus, measuring the OD of the supernatant after centrifugation can provide an estimation of the bacterial growth in the cocultures.To do this, the percentages of precipitated cells of each monoculture were calculated at different forces (from 100 to 500 x g) and times (1 and 2 min) using the measured OD before (A BC ) and after (A AC ) the centrifugation.Centrifugation at 200 x g for 1 min led to 87.9% of Chlamydomonas sedimentation, while only 2.1% of the bacterial cells dropped (meaning that 97.9% of the S. goyi cells remained in the supernatant).These parameters were chosen as a good compromise for SCS and used to evaluate the contribution of the bacteria to the OD in cocultures ( SCS OD 600 ).

Results
Identification of Stenotrophomonas goyi sp.nov.
A fortuitous contaminated Chlamydomonas reinhardtii culture (strain 704; CC-3597; https://www.chlamycollection.org/) was studied due to its enhanced hydrogen production capability.This alga culture turned out to be contaminated with three different bacterial strains (Fakhimi et al., 2023), one of them consisting in a white-pigmented bacterium (Figure 1).This bacterium was isolated after several rounds of plate streaking in TYM medium.First, partial PCR amplification and sequencing of the ribosomal 16S gene allowed the identification of this bacterium as a member of the Stenotrophomonas genus.Afterwards, the whole genome sequence was obtained.Genome assembling provided one single circular contig of 4,487,389 pb (Table 1).No plasmids or extrachromosomal elements were identified.
The RAST server identified 4,147 genes (4,066 CDS + 81 rRNAs and tRNAs) (Table 2).Out of these 4,066 CDS identified by RAST, 1,096 of them were in subsystems.Tentative genome annotation derived from the RAST server is available in Supplemental Table 1 as Extended data (González-Ballester et al., 2023).
Phylogenetic analyses were performed with both, the whole genome (Figure 2) and the inferred 16S rDNAs (Figure 3).Pairwise comparisons with the closest type strains genomes are shown in Table 3.These phylogenetic analyses revealed   Tree inferred with FastME 2.1.6.1 using the Genome BLAST Distance Phylogeny (GBDP) distances calculated from 16S rDNA gene sequences.The branch lengths are scaled in terms of GBDP distance formula d5.The numbers above branches are GBDP pseudo-bootstrap support values > 60% from 100 replications, with an average branch support of 78.6%.The tree was rooted at the midpoint.Branch lengths (black) and bootstraps (red) values are indicated.RNA16S lengths: 1,385-1,535 pb.Average δ statistics: 0.236 (Holland et al., 2002).Phylogenetic tree drawn with iTOL (Letunic and Bork, 2021).4).The addition of vitamins to the MM supplemented with glucose or lactose did not support the bacterium growth either (Table 4).However, the bacterium showed an excellent growth when cultivated in MM supplemented with yeast extract, tryptone, peptone or even Bovine Serum Albumin (BSA) (Table 4), suggesting that this bacterium has a great capacity to use peptides/amino acids as C source, and probably also as N source.Moreover, the peptides/amino acids could also provide, in addition to C and N sources, other essential nutrients or even palliate potential amino acids auxotrophies.Note that MM medium has sulfate as only S source.As commented before, the genome of S. goyi sp.nov. is lacking a functional sulfate assimilation pathway.Thereby S-containing amino acids, such as cysteine and methionine, could support the growth in medium rich in peptides/amino acids.

Closest strains to
To confirm this hypothesis, S. goyi sp.nov.was inoculated in plates of MM + glucose supplemented with different combinations of cysteine, methionine, biotin, and thiamine.Only plates containing cysteine and methionine supported the bacterial growth for several culturing rounds (Figure 4).This result confirms the cysteine and methionine growth dependence of S. goyi.sp.nov.Cysteine and methionine could provide either a reduced S source or complement an auxotrophy for these two amino acids.Since S. goyi sp.nov.genome has complete pathways for all the essential amino acids, is more likely that cysteine and methionine are being used as reduced S sources.Similar results were found for M. forte, where cysteine and methionine are required as S sources (Fakhimi et al., 2023a).

Sucrose
These results indicate that S. goyi sp.nov.and C. reinhardtii can establish a mutualistic when incubated in sugars-containing media.On the one hand, S. goyi sp.nov.can greatly support the growth of the C. reinhardtii in media supplemented with glucose or mannitol, which are two carbon sources that the alga cannot utilize.Likely, this growth promotion is due to the release of acetate and/or CO 2 from the bacteria after the sugar fermentation.Acetate is the sole organic carbon source that C. reinhardtii can utilize during heterotrophic/mixotrophic growth (Chaiboonchoe et al., 2014).On the other hand, S. goyi, sp.nov.can grow in media without amino acids/peptides supplementation when cocultured with C. reinhardtii, suggesting that the alga must provide some essential nutrients for the bacterium.Reduced S forms excreted by the alga (e.g., cysteine or methionine) could potentially explain the bacterium growth in the consortium.

Discussion and conclusions
Stenotrophomonas spp.are common constituents of the rhizosphere, and their potential for agricultural biotechnology is arising.However, their association with algae is poorly explored.Most plant growth-promoting bacteria (PGPB) are firstly identified in the rhizosphere and in association with plants.However, many PGPB are then also often commonly found in association with algae.This is likely reflecting that the kind of relationships established between bacteria and plants are similar to the relationships between bacteria and algae.This could potentially be the case for Stenotrophomonas spp., although the relative poor taxonomic curation and heterogeneity of genus may prevent the tracking of its association with algae.Some Stenotrophomonas spp.show a limited nutritional range, while others are capable of metabolic versatility (Ryan et al., 2009).S. goyi sp.nov. is unable to grow in the absence of a source of peptides/amino acids, which imply that in natural ecosystems it may rely on other microorganisms to obtain essential nutrients.As stated before, S. goyi sp.nov. is unable to use sulfate as S source.The peptides/amino acids are likely providing S-reduced forms (such as cysteine and methionine) to S. goyi sp.nov.
Stenotrophomonas goyi sp.nov.was isolated from an alga culture (C. reinhardtii) that showed an enhanced capacity to produce hydrogen and biomass when incubated in mannitol and yeast extract containing medium (Fakhimi et al., 2023a).This algal culture was simultaneously contaminated with two other bacteria: Microbacterium forte and Bacillus cereus.
Out of the three bacteria, M. forte was the main responsible for the enhanced algal hydrogen production.However, C. reinhardtii-M.forte cocultures were unable to produce hydrogen and biomass concomitantly.In addition to M. forte, the presence of S. goyi sp.nov.and B. cereus in the cocultures was needed to produce jointly hydrogen and algal biomass (Fakhimi et al., 2023a), which stresses the biotechnological interest of S. goyi sp.nov.
M. forte showed auxotrophy for biotin and thiamine, and like S. goyi sp.nov.was unable to grow on inorganic S sources (Fakhimi et al., 2023a).In this multispecies association, S. goyi sp.nov.and C. reinhardtii could alleviate the auxotrophy of M. forte sp.nov.for biotin and thiamine.S. goyi sp.nov.could also provide ammonium derived from the mineralization of the amino acids to the alga.On the other hand, the alga could provide S-reduced sources such as cysteine and methionine for S. goyi sp.nov.and M. forte.In any case, this multispecies association was mutually beneficial and prevented an excessive bacterial growth in cocultures, which could be one of the main drawbacks when algae-bacteria cocultures are used for biotechnological applications.
Nevertheless, the precise metabolic relationships established in this multispecies consortium that led to the extension of the C. reinhardtii cells viability during hydrogen production condition is not yet unraveled and need to be further investigated.
This project contains the following extended data: -Supplemental The conclusions are correct, the application of multispecies consortia including bacteria and green algae is a promising way for biological hydrogen production in an economically viably manner.

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound?Yes

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate?Yes Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results?
Functional annotation of the genome of the new bacteria reveled that some important pathways are not present in S. goyi genome.This data, beside several growth test carried out with aminoacids and /or peptide hydrolysates make the authors suggest that peptides and amino acids are a good C source, and probably also a good N or S source, for S. goyi, which can not grow in minimal medium not supplemented with aminoacids or peptides.Addition of aminoacid/peptides or co-cultivation with the microalga is necessary for the growth of this bacterial.Moreover, the microalga growth rate is practically doubled when cultured in the presence of the bacteria.The authors propose the establishment of a mutualistic relationship between C. reinhardtii and the bacteria, in which the bacteria would provide a carbon source (acetate or CO2 obtained from bacterial sugar fermentation) to the microalgae and the bacteria would obtain S-aminoacids excreted by the alga.
The authors suggest that many microalgal-associated bacteria can promote higher plant growth, being the characterization of this algal-bacterial consortium of interest to identify potent plant growth-promoting bacteria.

Suggestions:
The assessment of the bacterial growth by determination of optical density is adequate for pure cultures, but it is difficult to carry out in mixed algal-bacterial cultures.The authors have optimized and validated a selective centrifugal sedimentation approach to separate bacteria and microalgae.Although the approach seems to work well, I would suggest determination of colony forming units (CFU) to follow bacterial growth in future occasions.
In may experience, the sedimentation properties of the microalga can change along the growth cycle

Comments on this article Version 2
Reader Comment 22 Nov 2023 Tiago Henriques, University of Coimbra, Coimbra, Portugal In the "Data availability" section, "NCBI Gene" has the accession number CP116871 which directs to "Microbacterium sp.A(2022) strain A chromosome, complete genome".I think the accession nummber that should be there is CP124620.
Competing Interests: No competing interests were disclosed.
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Table 3 .
Pairwise dDDH values between S. goyi sp.nov.and the closest type strains genomes.The digital DNA-DNA Hybridization (dDDH) values are provided along with their confidence intervals (C.I.) for the three different Genome BLAST Distance Phylogeny (GBDP) formulas: a) formula d0: length of all High-Scoring segment Pairs (HSPs) divided by total genome length; b) formula d4: sum of all identities found in HSPs divided by overall HSP length; formula d6: sum of all identities found in HSPs divided by total genome length (Meier-Kolthoff et al., 2013).

Table 1 .
Main de novo sequencing and assembly statistics of Stenotrophomonas goyi sp.nov.genome.
including assimilation of nitrate (the whole assimilatory pathway is missing including nitrate transporters) and sulfate (only sulfite reductase is present).On the other hand, putative complete pathways for the glyoxylate cycle and biosynthesis of biotin, coenzyme A, pantothenate, riboflavin, tetrahydrofolate, glutathione, pyridoxal-P, lipoic acid, dTDP-L-rhamnose, UDP-N-acetyl-D-glucosamine, C5 isoprenoids, bacterial lipopolysaccharides, and antimicrobial proteins, among others, were present.Incomplete pathways for the degradation of aromatic compounds (including phenol, toluene, xylene, methylnaphthalene, 3-hydroxytoluene, and terephthalate) and myo-inositol biosynthesis, were also present.

Table 5 .
Growth of S. goyi sp.nov. at different temperatures and pHs.Lysogeny broth (LB) medium was used in all the conditions.

Table 1 [
Tentative annotation of the S. goyi sp.nov.genome]

the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Not applicable Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Biochemistry and Biotechnology of microalgae I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
Indicate in the legend of Figure1, that shows a plate with Chlamydomonas reinhardtii and Stenotrophomonas goyi sp.nov., the culture medium used.Is it bacterial or algal growth médium?Is it MM or has been supplemented with sugars?